According to the remarkable development of recombinant DNA technology, it has become possible to produce a large amount of useful proteins in E. coli, yeast, fungus, plant, animal and insect cells medically and industrially, where it could be obtained at only small amounts in nature. For example, proteins, such as interferon, interleukin 2, colony-stimulating factors, growth hormone, insulin-like growth factors and human serum albumin have been successfully produced by recombinant E. coli (Lee, S Y, Trends Biotechnol., 14:98, 1996). Particularly, technology for high cell-density culture of E. coli is well established whereby target proteins can be mass-produced at high productivity so that the cost of target protein production can be reduced. However, even though useful proteins are produced at a large amount by an efficient expression vector system, final yields of proteins are rapidly decreased, if a system for isolating and purifying protein is not well established. Such steps for separation and purification become much more significant when it is difficult to synthesize proteins, much time and effort are required, or the amount of protein production itself is small.
Steps for Separating and Purifying Proteins Generally Comprise as Follows:
(1) Cell extraction: cells or tissues are disrupted by glass homogenizer, mixer, polytron and sonicator, and so on, and then separated by a centrifugation; (2) Solubilization: when insoluble proteins are separated and purified, first of all, proteins should be solubilized. Various sorts of surfactants (SDS, Triton X-100, Nonidet P-40, CHAPS etc.) are used as an agent to solubilize a biological membrane protein; (3) Separation, condensation and dialysis of proteins: an ammonium sulfate precipitation by protein solubility, a molecular weight cut off method by the difference of protein molecular weight, a dialysis method by porous cellulose membrane, etc. are used before protein sample is purified; and (4) Final purification of proteins: since methods for purifying protein are various as shown in the Table 1 below, purification should be performed by combining methods according to the object of experiment and the kind of material. In general, in initial step, a method having high process capacity is selected even though separation activity is somewhat low, and as final step approaches, a method having high separation activity is used.
TABLE 1Typical methods for purifying proteinMethodFeatureKindColumnSeparation by pressureLPLC (low pressure liquidchromatography(or flux)chromatography)Separation of a bulk of proteinHPLC (high pressurechromatography)AffinityChromatography using affinityAffinity chromatography by antibodychromatographybetween a certain materialcolumn(ligand) and a proteinAffinity chromatography of GSTHigh purity and recovery ratefusion protein by glutathione columnProtein-specific affinitychromatographyOtherSeparation by protein chargeIon-exchange chromatographychromatography(isoelectric point), physiologicalGel filtrationactivity (chemical reactivity),Reverse-phase chromatographymolecular weight,Hydrophobic chromatographyhydrophobicityElectrophoresisSeparation by high resolutionIsoelectric focusingelectrophoresisSDS polyacrylamide gel electrophoresisTwo dimensional gel electrophoresis
To prevent proteolysis by protease, protease inhibitor can be mixed and used. For example, 0.4˜4.0 mM Petabloc SC (AEBSF; (4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride), 0.1˜1.0 mM PMSF (phenylmethyl sulphonyl fluoride) and 5˜50 mM Leupeptin are used against neutral serine proteases, 0.5˜5 mM EDTA against neutral metalloproteases, 0.1˜30 mM E-64 against cysteine proteases, and 1 mM Pepstain against aspartic acid proteases, are used, also a cocktail mixed with various inhibitors is generally used.
However, proteolysis can not be completely inhibited even with use of protease inhibitors. Particularly, when target proteins are separated and purified in plant extracts, or animal organs such as pancreas, stomach, liver or spleen in which various proteases are rich, or target proteins themselves are easily attacked by proteases, the loss of proteins is very severe.
Meanwhile, sHSPs are heat shock proteins (HSPs) with a low molecular weight of 12-43 kDa, which are induced by stress, such as heat shock or the overproduction of certain proteins. One or more of the sHSPs are present in all organisms from eukaryotes to prokaryotes, and the sHSPs known till now are given in the Table 2 below.
ATP-independent sHPSs perform the function of preventing protein aggregation irreversibly by binding with protein denatured under heat stress, and cooperate with ATP-dependent heat shock proteins to make denatured proteins refold correctly thereby, returning the denatured proteins to original state. For example, Kitagawa et al. have reported that IbpA and IbpB derived from E. coli prevent inactivation of citrate synthase by heat or oxidants (Kitagawa et al., Eur. J. Biochem., 269:2907, 2002), and Lee et al. have reported that HSP18.1 derived from pea prevents aggregation of denatured proteins, such as malate dehydrogenase (MDH), glyceraldehydes-3-phosphate dehydrogenase under heat stress (Lee et al., EMBO J., 16:659, 1997). Horwitz et al., have reported that α-crystallin derived from human prevents protein aggregation so as to help correct refolding of target proteins in dialysis process of denatured target proteins (Horwitz et al., Proc. Natl. Acad. Sci. USA, 89:10449, 1992). It was reported that sHSPs derived from Badyrbizobium japonicum prevent citrate synthase aggregation by heat (Studer and Narberhaus, J. Biol. Chem., 275:37212, 2000). It was reported that since Pfu-sHSP purified in an organism, which is stable under heat, protects proteins of cells under heat stress (WO 01/79250 A1), it stabilized Taq-polymerase and other enzymes at high temperature during PCR. Also, Ehrnsperger et al. have reported that sHSP 25 derived from Murine stabilizes proteins or peptides which are unstable in diagnostic assay (Ehrnsperger et al., Anal. Biochem., 259:218. 1998).
Such sHSPs have a conserved region in evolutionary processes and thus has been performing substantially similar functions. The present inventors have filed an application for a patent regarding composition for protecting protein degradation containing the sHSPs and a method of two-dimensional gel electrophoresis using the sHSPs (PCT/KR03/02539). However, there has not yet been any report on efficient prevention of target protein degradation by proteases in a cultivation process for preparing target proteins, a process for separating and purifying target proteins, and a reaction process using whole cell enzymes or partly purified enzymes.
TABLE 2The sHSPs familyOriginsHSPsAgrobacterium tumefaciens str.IbpAC58 (U. Washington)Arabidopsis thalianasHSPsBradyrbizobium japonicumHSPB, HSPH,HSPC, HSPFBrucella suis 1330IbpABuchnera aphidicola plasmidsHSPspBPS1Buchnera aphidicola str. APSIbpA(Acyrthosiphon pisum)Citrus tristeza virussHSPsEscherichia coli CFT073IbpA, IbpBEscherichia coli K12IbpA, IbpBEscherichia coli O157: H7IbpA, IbpBEDL933Escherichia coli O157: H7IbpA, IbpBHelicobacter pylori 26695IbpBHumanHSP27, α,β-crystallinMethanococcus jannaschiiHSP16.5Methanopyrus kandleri AV19IbpAMurineHSP25Mycobacterium leprae strain TNsHSPsMycobacterium tuberculosisHSP16.3Pirellula sp.IbpBPisum sativum (pea)HSP18.1Plasmodium falciparum 3D7sHSPsPseudomonas aeruginosa PA01IbpAPseudomonas putida KT2440IbpASaccharomyces cerevisiaeHSP26Salmonella enterica subsp.IbpA, IbpBenterica serovar TyphiSalmonella typhimurium LT2IbpA, IbpBShewanella oneidensis MR-1IbpAShigella flexneri 2a str. 2457TIbpA, IbpBShigella flexneri 2a str. 301IbpA, IbpBSinorhizobium meliloti 1021IbpASinorhizobium melilotiIbpAplasmid pSymAStreptococcus pyogenesIbpAStreptomyces coelicolor A3(2)sHSPsSulfolobus solfataricussHSPsSynechococcus vulcanusHSP16ThermoanaerobacterIbpAtengcongensis strain MB4TThermoplasma acidophilumIbpAYersinia pestis KIMsHSPs, IbpA,IbpBYersinia pestis strain CO92IbpA, IbpB
Accordingly, the present inventors have conducted intensive studies to develop a method for preventing target protein degradation by proteases in a cultivation process for preparing target proteins, a process for separation and purification, and an enzyme reaction using a target protein as biocatalyst, and consequently, found that the loss of target proteins by proteases can be prevented when the sHSPs are used, thereby completing the present invention.